Direct Identification Research Articles

Cell affinity screening combined with nanoLC-MS/MS based peptidomics for identifying cancer cell binding peptides from Bufo Bufo gargarizans

Animal venoms contain many peptides with high specificity and selectivity against their protein targets, a characteristic which makes venoms an invaluable source of potential drugs. High-sensitivity mass spectrometry (MS)- based peptidomic platform has evolved as a predominant method for natural peptide drug discovery due to its strength for direct and rapid identification of peptides and peptide-associated post-translational modifications (PTMs). In this study, we used cell-affinity assays combined with nanoLC-MS/MS based peptidomics to identify cancer cell binding peptides (CBPs) from Bufo Bufo gargarizans. We identified 76 potential cell binding peptides and 237 non-affinity peptides in venom extracts from Asiatic toads, and some were verified with MS-parallel reaction monitoring (PRM) mode. These peptides were further analyzed and internalized within human cells and some demonstrated anti-tumor properties in vitro. These specific peptides might be used as templates for peptide-based drug design or optimization.

The first isolation and detection of Ornithobacterium rhinotracheale from swollen head syndrome-infected broiler flocks in Iraq

Background and Aim:The swollen head syndrome (SHS) makes up complex diseases that infect the upper respiratory tract in poultry and causes several economic losses. Furthermore, this syndrome is considered one of the multifactorial etiological agents. Therefore, this study isolated and molecularly detected Ornithobacterium rhinotracheale (ORT) in poultry.Materials and Methods:This study was conducted at 67 broiler farms that had birds observed to be infected with the SHS from September 2018 until August 2019. Subsequently, swabs were collected from their trachea, infraorbital sinuses, and lungs, after which obtained samples were treated through two methods: (a) The direct method, by uploading samples on FTA cards, and the indirect method using a transport media. Afterward, reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the directly treated samples; howeverAQ1, the culture method, followed by PCR, was used to analyze the indirectly treated samples. Next, a partial 16S RNA gene was isolated using four positive PCR products, after which the effect of 16 antibiotics was studied on the seven local ORT strains isolated.Results:The quantity of ORT isolated using the direct method was 28 (41.7%) samples, which were all positive for the strain. Identification was by direct molecular identification (RT-PCR) from samples loaded on FTA cards. Alternatively, 7 (10.4%) ORTs were detected from the indirect method, as obtained using the culture method and biochemical tests. Then, PCR was subsequently used to confirm the results. As observed, 784 bp bands were shown for all seven ORT isolates. Furthermore, results revealed a significant difference in the detection of ORT strains between direct and indirect methods, with p-value (<0.05) and standard deviation of the error±0.038 for the direct, then ±0.061 for the indirect method. For further analysis on the strain types, four 784 bp PCR products were taken, then partial 16S ribosomal sequence typing was conducted. All these four strains were found to be recorded in NCBI for the 1st time as a local Iraqi strain, with accession numbers (MN931657, MN931656, MN931655, and MN931654). Notably, results also showed that all isolated strains were multidrug-resistant.Conclusion:From the results, ORT is proposed to be implicated as one of the etiological factors that cause SHSs in poultry. Phylogenetic analysis of the current ORT bacterial strains also showed that they are closely related to the Egyptian isolates.

Direct Target Site Identification of a Sulfonyl-Triazole Covalent Kinase Probe by LC-MS Chemical Proteomics.

Chemical proteomics is widely used for the global investigation of protein activity and binding of small molecule ligands. Covalent probe binding and inhibition are assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to gain molecular information on targeted proteins and probe-modified sites. The identification of amino acid sites modified by large complex probes, however, is particularly challenging because of the increased size, hydrophobicity, and charge state of peptides derived from modified proteins. These studies are important for direct evaluation of proteome-wide selectivity of inhibitor scaffolds used to develop targeted covalent inhibitors. Here, we disclose reverse-phase chromatography and MS dissociation conditions tailored for binding site identification using a clickable covalent kinase inhibitor containing a sulfonyl-triazole reactive group (KY-26). We applied this LC-MS/MS strategy to identify tyrosine and lysine sites modified by KY-26 in functional sites of kinases and other ATP-/NAD-binding proteins (>65 in total) in live cells. Our studies revealed key bioanalytical conditions to guide future chemical proteomic workflows for direct target site identification of complex irreversible probes and inhibitors.

Semantic enrichment on large scanned collections through their “satellite texts”: the paradigm of Migne’s Patrologia Graeca

PurposeThis paper aims to present a methodology for the semantic enrichment on the scanned collection of Migne’s Patrologia Graeca (PG), attempting to easily locate on the Web domain the scanned PG source, when a reference of this source is described and commented on another scanned or textual document, and to semantically enrich PG through related scanned or textual documents named “satellite texts” published by third people. The present enrichment of PG uses as satellite texts the Dorotheos Scholarios's Synoptic Index (DSSI) which act as metadata for PG.Design/methodology/approachThe methodology consists of two parts. The first part addresses the DSSI transcription via a proper web tool. The second part is divided into two subsections: the accomplishment of interlinking the printed column numbers of each scanned PG page with its actual filename, which is the build of a matching function, and the build of a web interface for PG, based on the generated Uniform Resource Identifiers (URIs) of the above first subsection.FindingsThe result of the implemented methodology is a Web portal, capable of providing server-less search of topics with direct (single click) navigation to sources. The produced system is static, scalable, easy to be managed and requires minimal cost to be completed and maintained. The produced data sets of transcribed DSSI and the JavaScript Object Notation (JSON) matching functions are available for personal use of students and scholars under Creative Commons license (CC-BY-NC-SA).Social implicationsScholars or anyone interested in a particular subject can easily locate topics in PG and reference them, using URIs that are easy to remember. This fact contributes significantly to the related scientific dialogue.Originality/valueThe methodology uses the transcribed satellite texts of DSSI, which act as metadata for PG, to semantically enrich PG collection. Furthermore, the built PG Web interface can be used by other satellite texts as a reference basis to further enrich PG, as it provides a direct identification of sources. The presented methodology is general and can be applied to any scanned collection using its own satellite texts.

Direct identification and visualisation of real-world contaminating microplastics using Raman spectral mapping with multivariate curve resolution-alternating least squares

Microplastics (MPs) contamination is ubiquitous in environmental matrices worldwide. Moreover these pollutants can be ingested by organisms and transported to organs via the circulatory system. Although efficient methods for the analysis of MPs derived from environment matrices and organisms’ tissue samples have been developed after special sample pre-treatment, there remains a need for an optimised approach allowing direct identification and visualisation these MPs in real environmental matrices and organismal samples. Herein, we firstly used a multivariate curve resolution-alternating least squares (MCR-ALS) analysis of Raman hyperspectral imaging data to direct identification and visualisation of MPs in a complex serum background. Four common MPs types including polyethylene (PE), polystyrene (PS), polypropylene (PP) and polyethylene terephthalate (PET) were identified and visualised either individually or in mixtures within spiked samples at an 8-μm spatial resolution. Moreover, Raman imaging based on MCR-ALS was successfully applied in fish faeces biological samples and environmental sand samples for in situ MPs identification directly without washing or removal of organic matter. The current results demonstrate Raman imaging based on MCR-ALS as a novel imaging approach for direct identification and visualisation of MPs, through extraction of MPs’ chemical spectra within a complicated biological or environmental background whilst eliminating overlapping Raman bands and fluorescence interference.

P–769 FTIR spectroscopy analysis reveals differences between human embryo culture media composition by type of formulation and by manufacturer

Abstract Study question Can we detect the variation in different commercial human embryo culture media composition by Fourier-transform infrared (FTIR) spectroscopic analysis? Summary answer The spectra reveals distint features that allow distinguishing between continuous and sequential media, as well as between manufacturers of the same media type. What is known already: Manufacturers do not fully disclose commercially available culture media formulations. For this reason, it is important to gain insight into de differences between the available formulations, in order to understand how they might be linked with efficacy and safety ART parameters. Fourier Transform Infra-Red (FTIR) can be used for this purpose. Study design, size, duration Culture media samples (1 mL) were collected from local IVF laboratories, and stored frozen at –20 °C. Five repeats of 25 µL aliquots from the samples were analysed using FTIR spectroscopy to acquire the whole molecular fingerprint of each culture media. Participants/materials, setting, methods Three continuous (SAGE 1-STEP, Origio, G-TL, Vitrolife, and GERI, Genea) and four sequential (G1 PLUS, Vitrolife; Sequential Cleav with phenolred, Origio; G2 PLUS, Vitrolife and Sequential Blast with phenolred, Origio) media were analysed. Different pre-processing methods (atmospheric and baseline correction, normalizations, and derivatives) were carried out to minimize physical artefacts while highlighting chemical features. To compare the spectra of different media, multivariate analysis, as principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed. Main results and the role of chance The whole molecular fingerprint of all media analysed showed a similar pattern, revealing that, overall, the composition is very similar. However, PCA and HCA analysis revealed that significant differences exist, both between media type (continuous vs. sequential), and between different manufacturers within the same media type. Average linkage clustering using Spearman’s rank correlation confirms the similarities between the continuous and the sequential formulations. An analysis focusing the fingerprint region of the spectra (900 – 1700 cm^–1), also revealed variability between manufacturer, between media type (continuous vs. sequential) and of stage-specific media (cleavage vs. blastocyst). For instance, GERI media visually appeared to have distinct peaks compared to all other media, which was confirmed later through multivariate statistical analysis. Limitations, reasons for caution FTIR spectroscopy does not allow for a direct identification of the analytes present in the culture media, we can only infer the functional groups, but that are common on diverse biomolecules. Wider implications of the findings: FTIR analysis reveals differences between different media, such as cleavage and blastocyst specific, sequential and continuous, or manufacturer’s formulations. The increased resolution of the FTIR profile proves to be a powerful tool for analysing human embryo media, and could be used to establish correlations with media clinical performance and safety. Trial registration number Not applicable

Misdiagnosed tuberculosis being corrected as Nocardia farcinica infection by metagenomic sequencing: a case report

BackgroundDisseminated nocardiosis is liable to be misdiagnosed owing to the non-specific clinical manifestations and laboratory/imaging findings. Metagenomic next-generation sequencing (mNGS) is a culture-independent and rapid method for direct identification of all microorganisms in clinical specimens.Case presentationA 72-year-old man was admitted to our hospital on February 20, 2019 with a history of recurrent cough, expectoration, fever, and diarrhea since 1 month, and unconsciousness since 1 week. Contrast-enhanced magnetic resonance imaging of head showed multiple lesions in the bilateral cerebral hemispheres, brainstem, and cerebellar hemispheres. The presumptive diagnosis was disseminated tuberculosis, although all tests for mycobacterium were negative. However, the patient did not benefit from antituberculosis treatment. Repeat MRI showed multiple abnormal signals in the brain and progression of meningeal thickening. Cerebrospinal fluid and bronchoalveolar lavage fluid specimens were subsequently sent for PMSeq metagenomics sequencing; the results indicated Nocardia. farcinica as the predominant pathogen. The anti-TB treatment was stopped and the patient was prescribed sulphamethoxazole in combination with linezolid and meropenem for nocardiosis. He showed gradual neurological improvement and was transferred to Huashan Hospital. He was discharged from the hospital on April 19, 2019, but died of persistent diarrhea on May 26, 2019.ConclusionsPatients with suspected nocardiosis do not always respond to conventional treatment; therefore, mNGS can facilitate diagnosis and timely treatment decision-making.

Direct identification of HLA-presented CD8 T cell epitopes from transmitted founder HIV-1 variants.

Cytotoxic T lymphocytes (CTLs) are a critical arm of the immune response to viral infections. The activation and expansion of antigen specific CTL requires recognition of peptide antigens presented on class I major histocompatibility complex molecules (MHC-1) of infected cells. Methods to identify presented peptide antigens that do not rely on the pre-existence of antigen specific CTL are critical to the development of new vaccines. We infected activated CD4+ T cells with two HIV-1 transmitted founder (TF) isolates and used high-resolution mass spectrometry (MS) to identify HIV peptides bound on MHC-1. Using this approach, we identified 14 MHC-1 bound peptides from across the two TF isolates. Assessment of predicted binding thresholds revealed good association of the identified peptides to the shared HLA alleles between the HIV+ donors and the naïve PBMC sample with three peptides identified through peptide sequencing inducing a CD8 T-cell response (p < 0.05). Direct infection of naïve CD4 cells by HIV TF isolates and sequencing of MHC-I presented peptides by HPLC-MS/MS enables identification of novel peptides that may be missed by alternative epitope mapping strategies and can provide valuable insight in to the first peptides presented by an HIV-infected CD4 cell in the first few days post infection.

Development of pteridophyte catarium (herbarium catalog) media in plantae materials for X graders of SMA Islam Batu

: Plantae material is one of the materials that requires a direct observation process to determine the characteristics, morphology, metagenesis, and benefits for the environment, especially in Pteridophyte material. So far, students only use textbooks and students have not been invited to learn with contextual learning objects. Thus, media is needed to facilitate direct observation and identification of plants. The purpose of this research is to produce learning media Pteridophyte catarium (Catalog Herbarium). This type of research is research and development (R&amp;D) with the ADDIE development model. The results showed that the quality of the product according to media experts was very feasible with an assessment percentage of 77.08%, as well as according to material experts who said it was very feasible with a 95% rating percentage. The results of the teacher's response were 79.16% and the results of readability by students on a small and large scale respectively were 76.5% and 81.16%, indicating very decent quality. It was concluded that the product in the form of Pteridophyte catarium learning media was very suitable to be used as a teacher's learning media in plantae biology learning activities.

Nanopore Sequencing Accurately Identifies the Cisplatin Adduct on DNA.

Cisplatin, which selectively binds to N7 atoms of purines to inhibit normal replication and transcription, is a widely applied chemotherapeutic drug in the treatment of cancer. Though direct identification of cisplatin lesions on DNA is of great significance, existing sequencing methods have issues such as complications of preamplification or enrichment-induced false-positive reports. Direct identification of cisplatin lesions by nanopore sequencing (NPS) is in principle feasible. However, relevant investigations have never been reported. By constructing model sequences (83 nucleotides in length) containing a sole cisplatin lesion, identification of corresponding lesions by NPS is achieved with <10 ng of input sequencing library. Moreover, characteristic high-frequency noises caused by cisplatin lesions are consistently observed during NPS, clearly identifiable in corresponding high-pass filtered traces. This feature is, however, never observed in any other combinations of natural DNA bases and could be taken as a reference to identify cisplatin lesions on DNA. Further investigations demonstrate that cisplatin stalls the replication of phi29 DNA polymerase, which appears as a ∼5 pA level fluctuation in the single-molecule resolution. These results have confirmed the feasibility of NPS to identify cisplatin lesions at the genomic level and may provide new insights into understanding the molecular mechanism of platinum-based drugs.

In situ element analysis of spodumenes by fs-LA-ICPMS with non-matrix-matched calibration: Signal beat and accuracy

In this work, in situ laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) analysis with non-matrix-matched calibration is proposed for determining the composition of spodumene samples. Accuracy of the quantification in dependence on instrument operating conditions and the signal beat effect on quantification accuracy have been investigated in detail. Using NIST SRM 610 as reference material for external calibration, the mass fractions of major, minor and trace elements (Li, Si, Al, Na, Mg, Mn, Fe, Ga, and others) were determined using femtosecond-LA-ICPMS and compared with nanosecond-LA-ICPMS. Helium (He) was employed as the carrier gas in the ablation cell, and the aerosols produced under hole drilling ablation condition are then transported to a quadrupole ICPMS by a mixture of He and Ar as the make-up gas. Results show that the carrier gas flow rate had a substantial effect on both transient signal structure and LA-ICPMS quantification. With different He flow rates, both synchronized and non-synchronized acquisition with and without aliasing in the transient signals recorded were compared. Synchronized acquisition settings could attenuate the oscillations in the transient signals substantially but showed large variability (up to 18% RSD for Li2O) among individual acquisitions. Non-synchronized acquisition, with and without visible aliasing, on the other hand, was producing reproducible results without systematic biases when averaging ion signals from a sufficient number of scans (>100) for quantification. For the latter it was found that non-aliasing methods allowed for a direct identification of heterogeneities or inclusions in the transient signals and these were thus preferred for analysis. The accuracy of the analyses with non-matrix matched calibration was evaluated by comparing the matrix Al2O3/SiO2 mass fraction ratios with those obtained by electron probe microanalysis (EPMA). It was found that operation with only 0.2 L/min of He achieved the best agreement, while higher gas flow rates caused an increase in Al2O3/SiO2 and Li2O/SiO2 mass fraction ratios. This is considered to be a result of differences in ionization efficiency within the ICP caused by the higher abundance of easily ionized elements in the spodumenes compared to the calibration standard. By selecting appropriate acquisition settings for sequential MS detection and robust operating conditions, the repeatability of the analyses could be improved to values less than 1% for the Li2O mass fractions determined. When using the proposed settings for the analysis of 11 spodumene samples of different origin, the Al2O3/SiO2 mass fraction ratios agreed within −2% to +4% to those obtained by EPMA. These results confirm that spodumenes can be accurately analyzed in situ by LA-ICPMS without matrix-matched calibration standards. While matrix elements of the studied spodumenes were all in a comparable range, minor and trace elements showed substantial intra- and inter-sample variabilities.

Use of microRNA-encoded peptides to improve agronomic traits.

MicroRNA-encoded peptides (miPEPs) are small regulatory peptides encoded by primary transcripts of microRNAs (miRNAs). In plants, they activate the transcription of their cognate miRNA and external application of chemically synthetized miPEPs can lead to phenotypes suitable for agronomic applications. We developed an experimental pipeline to screen Arabidopsis thaliana miPEPs for root development and identified several miPEPs having positive and negative effect on root growth. Direct identification of A. thaliana miPEPs homologs in two species of agronomic interest showed that specific peptides can improve cabbage root development while others decrease weed root development. We showed here that simple external application of miPEPs by plant watering can directly improve crop growth or decrease weed development in a specie specific manner.

Coformulation with Tattoo Ink for Immunological Assessment of Vaccine Immunogenicity in the Draining Lymph Node

Characterization of germinal center B and T cell responses yields critical insights into vaccine immunogenicity. Nonhuman primates are a key preclinical animal model for human vaccine development, allowing both lymph node (LN) and circulating immune responses to be longitudinally sampled for correlates of vaccine efficacy. However, patterns of vaccine Ag drainage via the lymphatics after i.m. immunization can be stochastic, driving uneven deposition between lymphoid sites and between individual LN within larger clusters. To improve the accurate isolation of Ag-exposed LN during biopsies and necropsies, we developed and validated a method for coformulating candidate vaccines with tattoo ink in both mice and pigtail macaques. This method allowed for direct visual identification of vaccine-draining LN and evaluation of relevant Ag-specific B and T cell responses by flow cytometry. This approach is a significant advancement in improving the assessment of vaccine-induced immunity in highly relevant nonhuman primate models.

Electromechanical field effects in InAs/GaAs quantum dots based on continuum [formula omitted] and atomistic tight-binding methods

A comparison between k→·p→ and tight-binding methods for the analysis of InAs/GaAs quantum dot bandstructures is presented based on a fully coupled computation of electromechanical effects. Electromechanical effects are addressed using a continuum elastic model for the k→·p→ method and a pre-conditioned Valence Force Field algorithm for the tight-binding atomistic calculations. The Valence Force Field method allows the direct identification of the impact of internal strain. Results to ensure model parameter consistency between the two methods are also given by comparing bulk and unstrained quantum-well dispersion relations.The quantum dot size dependence of the bandstructure is investigated based on the models including electromechanical fields. Additionally, the effect of the electromechanical fields is studied for a specific dot size by comparing results with and without electromechanical fields. Good agreement is found for the confined energy levels but model differences show up in the symmetry of probability densities mainly due to the underlying crystal structure details taken into account by the tight-binding method but lacking in the k→·p→ formalism. The latter follows from not including bulk inversion-asymmetry effects in the k→·p→ method. Inclusion of piezoelectric field effects in the k→·p→ method, however, restores the correct symmetry in the k→·p→ model (in agreement with the tight-binding symmetry).Results are also given for oscillator strengths where both quantitative and qualitative differences are found in the comparison of k→·p→ and tight-binding models.

High throughput techniques for the rapid identification of electroactive microorganisms

Electroactive microorganisms (EAM), capable of executing extracellular electron transfer (EET) in/out of a cell, are employed in microbial electrochemical technologies (MET) and bioelectronics for harnessing electricity from wastewater, bioremediation and as biosensors. Thus, investigation on EAM is becoming a topic of interest for multidisciplinary areas, such as environmental science, energy and health sectors. Though, EAM are widespread in three domains of life, nevertheless, only a few hundred EAM have been identified so far and hence, the rapid identification of EAM is imperative. In this review, the techniques that are developed for the direct identification of EAM, such as azo dye and WO3 based techniques, dielectrophoresis, potentiostatic/galvanometric techniques, and other indirect methods, such as spectroscopy and molecular biology techniques, are highlighted with a special focus on time required for the detection of these EAM. The bottlenecks for identifying EAM and the knowledge gaps based on the present investigations are also discussed. Thus, this review is intended to encourage researchers for devolving high-throughput techniques for identifying EAM with more accuracy, while consuming less time.

Therapeutic Cancer Vaccination with Immunopeptidomics-Discovered Antigens Confers Protective Antitumor Efficacy.

Simple SummaryImmunotherapy has revolutionized cancer treatment, yet many tumors remain resistant to current immuno-oncology therapies. Here we explore a novel, customized oncolytic adenovirus vaccine platform as immunotherapy in a resistant tumor model. We present a workflow for customizing the oncolytic vaccine for improved tumor targeting. This targeting is based on experimentally discovered tumor antigens, which are incorporated as active components of the vaccine formulation. The pipeline may be further applied for designing personalized therapeutic cancer vaccines.Knowledge of clinically targetable tumor antigens is becoming vital for broader design and utility of therapeutic cancer vaccines. This information is obtained reliably by directly interrogating the MHC-I presented peptide ligands, the immunopeptidome, with state-of-the-art mass spectrometry. Our manuscript describes direct identification of novel tumor antigens for an aggressive triple-negative breast cancer model. Immunopeptidome profiling revealed 2481 unique antigens, among them a novel ERV antigen originating from an endogenous retrovirus element. The clinical benefit and tumor control potential of the identified tumor antigens and ERV antigen were studied in a preclinical model using two vaccine platforms and therapeutic settings. Prominent control of established tumors was achieved using an oncolytic adenovirus platform designed for flexible and specific tumor targeting, namely PeptiCRAd. Our study presents a pipeline integrating immunopeptidome analysis-driven antigen discovery with a therapeutic cancer vaccine platform for improved personalized oncolytic immunotherapy.

Challenges targeting cancer neoantigens in 2021: a systematic literature review

Introduction Cancer neoantigens represent important targets of cancer immunotherapy. The goal of cancer neoantigen vaccines is to induce neoantigen-specific immune responses and antitumor immunity, while minimizing the potential for autoimmune toxicity. Advances in sequencing technologies, neoantigen prediction algorithms and other technologies have dramatically improved the ability to identify and prioritize cancer neoantigens. Unfortunately, results from preclinical studies and early phase clinical trials highlight important challenges to the successful clinical translation of neoantigen cancer vaccines. Areas covered In this review, we provide an overview of current strategies for the identification and prioritization of cancer neoantigens with a particular emphasis on the two most common strategies used for neoantigen identification: (1) direct identification of peptide ligands eluted from peptide-MHC complexes, and (2) next-generation sequencing combined with neoantigen prediction algorithms. We highlight the limitations of current neoantigen prediction pipelines, and discuss broader challenges associated with cancer neoantigen vaccines including tumor purity/heterogeneity and the immunosuppressive tumor microenvironment. Expert Opinion Despite current limitations, neoantigen prediction is likely to improve rapidly based on advances in sequencing, machine-learning, and information sharing. The successful development of robust cancer neoantigen prediction strategies is likely to have significant impact, with the potential to facilitate cancer neoantigen vaccine design.

Urban foraging, resilience and food provisioning services provided by edible plants in interstitial urban spaces in Mexico City

ABSTRACT In the context of decreasing food security, and as part of the ‘100 Resilient Cities’ group, Mexico City (CDMX) has committed itself to achieving resilience through local food systems, green infrastructure and ecosystem services development. This paper reports on two surveys mapping the distribution of species of edible plants growing in publicly accessible interstitial spaces (e.g. central reservations and pavements) in subtropical, central CDMX. It also reports on an initial assessment of the feasibility of using such spaces for foraging. Rather than improving everyday food supplies, the maintenance of these spaces as areas for emergency foraging would ideally increase urban resilience in the face of extreme food supply shocks. The surveys used a direct species identification approach whilst walking ∼20 km of streets and identified thirty-six species of edible plants including not only fruits and herbs, but also vegetables. The results permitted the satisfactory assessment of three out of five feasibility conditions: (i) suitable growing conditions, (ii) provisioning capability and (iii) the existence of multiple plant dispersal pathways (including both natural processes and deliberate planting, as well as, more speculatively, unintentional human-mediated dispersal). Two other important feasibility conditions for foraging – (iv) usage and (v) a favourable planning environment – were not able to be judged based on the field data. CDMX promotes the planting of fruit trees in avenues, but this paper shows that local socio-ecological systems sustain a greater variety of edibles growing in a wider range of interstitial spaces. Ignoring this would represent a missed opportunity for urban resilience.

Abstract 1891: Targeted mass spectrometry provides direct evidence of a human endogenous retrovirus type E (HERV-E) antigen presented on the surface of clear cell renal cell carcinoma cells

Abstract Background: Previously, our group identified donor-derived T cells that mediated regression of metastatic clear cell renal cell carcinoma (ccRCC) in a patient who underwent an allogeneic hematopoietic stem cell transplant. A T cell clone isolated from this patient had direct cytotoxicity against ccRCC cells. Its target antigen was discovered to be encoded by a human endogenous retrovirus type E (named CT-RCC HERV-E) that had selective expression in ccRCC. Subsequent experiments showed this T cell clone recognized a cryptic 10-mer peptide called CT-RCC1 (ATFLGSLTWK), derived from a noncanonical open reading frame of the CT-RCC HERV-E and presented in the context of HLA-A11. This study aimed to establish a proteomic workflow using targeted parallel reaction monitoring (PRM) mass spectrometry (MS) for the direct identification of CT-RCC1 peptide in ccRCC HLA ligandomes and to use that platform to identify other CT-RCC HERV-E derived peptides presented in the context of more common HLA alleles such as HLA-A02. Methods: In this study, four ccRCC cell lines were used: HLA-A11 positive/CT-RCC HERV-E positive, HLA-A11 negative/CT-RCC HERV-E positive, HLA-A11 positive/CT-RCC HERV-E negative and HLA-A11 negative/CT-RCC HERV-E negative. Additionally, we used the HLA-A11 positive/CT-RCC HERV-E positive cell line to create both beta 2-microglobulin and CT-RCC HERV-E CRISPR knockout lines. HLA-peptide complexes were immunopurified from cell lysates with anti-HLA Class I antibodies cross-linked to Protein A Sepharose beads. Peptides were separated from HLA molecules using Sep-Pak tC18 reverse phase separation columns and subjected to targeted parallel reaction monitoring (PRM) mass spectrometry (MS) analysis. Synthetic CT-RCC1 peptide was used to generate optimum PRM transitions, determine the best collision energy, and obtain the full fragment ion spectrum. Data analysis was done using the Skyline package. Results: CT-RCC1 peptide was identified by targeted MS to be presented on the surface of HLA-A11 positive/CT-RCC HERV-E positive ccRCC cells but not in those same cells that had CT-RCC HERV-E or beta 2-microglobulin knocked out using CRISPR. Additionally, the CT-RCC1 peptide was not detected in cells which were naturally HLA-A11 negative, CT-RCC HERV-E negative, or both. Conclusions: Targeted MS identified the HERV-E derived HLA-A11 restricted peptide CT-RCC1 to be expressed on the surface of ccRCC cells. This study defines a reproducible immunopeptidomic workflow using targeted MS for identifying other antigens derived from this HERV-E that may be presented in the context of common HLA alleles. Acknowledgments: We acknowledge the NIH Intramural Research Program and Rancic O'Neill Renal Cell Cancer Research Fellowship Fund for supporting this project. Citation Format: Stefan Barisic, Elena Cherkasova, Yong Chen, Stephanie Pierre, Richard W. Childs. Targeted mass spectrometry provides direct evidence of a human endogenous retrovirus type E (HERV-E) antigen presented on the surface of clear cell renal cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1891.

Abstract 1895: caAtlas: An immunopeptidome atlas of human cancer

Abstract At the core of immunotherapy is the T cell recognition of tumor antigens. Thus, comprehensive characterization of tumor antigens is essential for the design of effective and safe cancer immunotherapy strategies. Mass spectrometry (MS)-based immunopeptidomics allows high-throughput, direct identification of major histocompatibility complex (MHC)-bound peptides in vivo. Here, we collected 42 published human immunopeptidomic datasets, which include 327 cancer samples covering nine cancer types and 216 non-cancerous samples. A systematic, quality-controlled analysis of all 51.6 million MS/MS spectra using an open search algorithm identified 364,283 peptides, including 11,647 modified peptides associated with 80 types of modifications. Unmodified peptides showed high levels of predicted binding affinities by NetMHCpan; however, the predicted binding affinities for the modified peptides were much lower, suggesting a deficiency of existing computational tools in making predictions for modified peptides. Analysis of the 416 phosphorylated and 1,239 acetylated HLA class I peptides revealed distinct sequence motifs, and the pattern for the phosphopeptides was consistent with previous reports. Further interrogation of all identified peptides revealed evidence for tumor specific-presentation of hundreds of known and novel cancer-testis antigens as well as tumor associated antigens. We also found non-tumor-specific presentation of 44 previously annotated cancer-testis antigens. Applying NeoQuery, a newly developed computational pipeline, to our dataset identified immunopeptidomic evidence for more than 1,000 somatic mutation-derived neoantigens, and a subset of them were further validated through a traditional MS/MS peptide identification pipeline. We make all these data together with annotated MS/MS spectra supporting each individual antigen identification in a publicly accessible, easily browsable web portal named cancer antigen atlas (caAtlas, http://www.zhang-lab.org/caatlas/). caAtlas provides a fundamental resource for the selection and prioritization of MHC-bound peptides for immunogenicity testing and cancer immunotherapy development. Citation Format: Xinpei Yi, Yuxing Liao, Bo Wen, Kai Li, Yongchao Dou, Sara R. Savage, Bing Zhang. caAtlas: An immunopeptidome atlas of human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1895.